The present invention relates to retrovirus strains of the HIV-1 group, group O, and in particular the strains called BCF02 (ESS), BCF01 (FAN), BCF06 (LOB), BCF07 (MAN), BCF08 (NKO), BCF11 (NAN) and BCF03 (POC), to fragments of the said retroviruses and to their applications as diagnostic reagent and as immunogenic agent.
Two distinct types of HIV (human immunodeficiency virus: HIV-1 and HIV-2) have been described and are the agents responsible for AIDS. Analysis of their nucleic acid sequence has made it possible to identify various subtypes of HIV-1, although no correlation could be established between variability and pathogenicity. Similarly, HIV-2 exhibits a greater genetic and biological diversity than that previously envisaged.
Analysis of nucleotide fragments of various HIV-1 isolates has shown the existence, through the analysis of the env gene, of at least 7 different subtypes, called A to G (MYERS G. et al., Human retroviruses and AIDS, 1993, Los Alamos Nat. Lab.).
More recently, two other isolates, considered to be considerably more distant from the other 7 subtypes, that is to say whose sequence homology is the most distant from that of the reference HIV-1 strains, have also been isolated: HIV-1ANT70 and HIV-1MVP5180, obtained from Cameroonian patients, and have been attached to a new HIV-1 group, group O, as opposed to group M corresponding to the 7 abovementioned A-G subtypes, taking into account their genomic organization (5' LTR Gag Pol Vif Vpu Vpr Tat Rev Env Nef LTR 3'), (Patent Application WO 89/12094, European Patent Application No. 0,591,914, GURTLER L. G. et al., J. Virol., 1994, 68, 1581-85).
Analysis of the DNA sequences has shown 65-70% similarity with HIV-1 and 56% with HIV-2.
By using a competition immunoblotting method, using a peptide V3 from MVP5180, a 7-8% prevalence is found in Yaounde. This prevalence might be under-estimated since the V3 loop is known, in all the HIV-1 subtypes, to be a highly variable region. Molecular studies also indicate the presence of group O virus in Gabon, France, Spain and Germany.
Ongoing serological studies reveal group O HIV-1 infections in Nigeria, Niger and Senegal.
Knowledge of these various groups and subtypes is particularly important for developing:
reagents for screening for HIV infections which are sufficiently sensitive and specific, that is to say which do not lead to false-negative or false-positive results; and PA1 compositions which protect against all existing subtypes, including the entire group O viruses. PA1 either one of the following sequences, included in the C2V3-env gene fragment (hypervariable loop of gp120): SEQ ID No. 1 (BCF02 (ESS)), SEQ ID No. 2 (BCF08 (NKO)), SEQ ID No. 3 (BCF03 (POC)), SEQ ID No. 4 (BCF06 (LOB)), SEQ ID No. 5 (BCF07 (MAN)), SEQ ID No. 6 (BCF01 (FAN)), SEQ ID No. 7 (BCF11 (NAN)), SEQ ID No. 50 (BCF09), SEQ ID No. 51 (BCF12), SEQ ID No. 52 (BCF13), SEQ ID No. 53 (BCF14), PA1 or one of the following sequences, included in the gp14 fragment of the env gene: SEQ ID No. 8 (BCF02 (ESS)), SEQ ID No. 9 (BCF08 (NKO)), SEQ ID No. 10 (BCF03 (POC)), SEQ ID No. 11 (BCF06 (LOB)), SEQ ID No. 12 (BCF07 (MAN)), SEQ ID No. 13 (BCF01 (FAN)), SEQ ID No. 14 (BCF11 (NAN)), SEQ ID No. 54 (BCF09), SEQ ID No. 55 (BCF12), SEQ ID No. 56 (BCF13), SEQ ID No. 57 (BCF14), PA1 or one of the following sequences, included in the gag gene: SEQ ID No. 15 (BCF02 (ESS)), SEQ ID No. 16 (BCF08 (NKO)), SEQ ID No. 17 (BCF03 (POC)), SEQ ID No. 18 (BCF05 (LOB)), SEQ ID No. 19 (BCF07 (MAN)), SEQ ID No. 20 (BCF01 (FAN)), SEQ ID No. 21 (BCF11 (NAN)), SEQ ID No. 58 (BCF09), SEQ ID No. 59 (BCF12), SEQ ID No. 60 (BCF13), SEQ ID No. 61 (BCF14), PA1 or, if the sequence is not identical to one of the above nucleotide sequences, or is not complementary to one of these sequences, is nonetheless capable of hybridizing with a nucleic sequence derived from a group O HIV-1 virus. PA1 * sequences gag PA1 * sequences gp41 PA1 sequences C2V3: PA1 a step of extracting the nucleic acid to be detected, belonging to the genome of the HIV-1 type virus, which may be present in the biological sample and, where appropriate, a step of treating the nucleic acid with the aid of a reverse transcriptase, if the latter is in RNA form, PA1 at least one cycle comprising the steps of denaturation of the nucleic acid, annealing with at least one sequence in accordance with the invention and extension of the hybrid formed, in the presence of the appropriate reagents (polymerizing agent such as DNA polymerase and dNTP), and PA1 a step of detecting the possible presence of the nucleic acid belonging to the genome of a group O HIV-1 type virus (group specificity). PA1 those expressed by the C2V3-env gene fragment in accordance with the invention: SEQ ID No. 26 (BCF02 (ESS)), SEQ ID No. 27 (BCF01 (FAN)), SEQ ID No. 28 (BCF01 (FAN)), SEQ ID No. 29 (BCF06 (LOB)), SEQ ID No. 30 (BCF07 (MAN)), SEQ ID No. 31 (BCF11 (NAN)), SEQ ID No. 32 (BCF08 (NKO)), SEQ ID No. 33 (BCF08 (NKO)), SEQ ID No. 34 (BCF03 (POC)), SEQ ID No. 35 (BCF03 (POC)), SEQ ID No. 62 (BCF09), SEQ ID No. 63 (BCF12), SEQ ID No. 64 (BCF13), SEQ ID No. 65 (BCF14), PA1 those expressed by the gp41 env gene fragment in accordance with the invention: SEQ ID No. 36 (BCF02 (ESS)), SEQ ID No. 37 (BCF01 (FAN)), SEQ ID No. 38 (BCF06 (LOB)), SEQ ID No. 39 (BCF07 (MAN)), SEQ ID No. 40 (BCF08 (NKO)), SEQ ID No. 41 (BCF03 (POC)), SEQ ID No. 42 (BCF11 (NAN)), SEQ ID No. 66 (BCF09), SEQ ID No. 67 (BCF12), SEQ ID No. 68 (BCF13), SEQ ID No. 69 (BCF14), PA1 those expressed by the gag gene fragment in accordance with the invention: SEQ ID No. 43 (BCF02 (ESS)), SEQ ID No. 44 (BCF01 (FAN)), SEQ ID No. 45 (BCF06 (LOB)), SEQ ID No. 46 (BCF07 MAN)), SEQ ID No. 47 (BCF11 (NAN)), SEQ ID No. 48 (BCF08 (NKO)), SEQ ID No. 49 (BCF03 (POC)), SEQ ID No. 70 (BCF09), SEQ ID No. 71 (BCF12), SEQ ID No. 72 (BCF13), SEQ ID No. 73 (BCF14).
Indeed, it has been shown, in particular, that some detection reagents were not sufficiently sensitive and did not always make it possible to detect group O HIV-1 infections (LOUSSERT-AJAKA I. et al., Lancet, 1994, 343, 1393-94), which has led to the withdrawal of three screening kits and to the declassification of two others on the French market.
The results show that the group O viruses are very distant from the group M HIV-1 subtypes. These results indicate that the HIV-1 viruses ought to be classified in two different groups, namely: HIV-1 M and HIV-1 O. The group O viruses appear to be capable of being transmitted by horizontal and vertical routes, leading to a very wide distribution of this infection. The pathogenicity of these viruses is under study. The divergence of these viruses should be taken into account in the sensitivity of diagnostic tests and in the development of vaccines.
Consequently, the Applicant set itself the objective of providing fragments, derived from selected group O HIV-1 strains, capable of allowing both detection of the entire group O HIV-1 viruses and specific intra-group O differentiation and which are also capable of inducing protection against the entire HIV-1 subtypes, including group O, which fragments make it possible to avoid obtaining false-negative or false-positive results; to do this, the inventors selected a set of strains and sequences, derived essentially from a region of the env gene, in particular at the level of a hypervariable fragment situated in the V3 loop (C2V3) or at the level of gp41 and of a region of the gag gene.
The subject of the present invention is group O HIV-1 strains exhibiting the morphological and immunological characteristics of one of the retroviruses deposited at the Collection Nationale de Cultures de Microorganismes held by Institut Pasteur under the numbers I-1544 (called BCF02 (ESS)), I-1543 (called BCF01 (FAN)), I-1546 (called BCF07 (MAN)), I-1547 (called BCF08 (NKO)), I-1545 (called BCF03 (POC)), on the date of Feb. 24, 1995.
The subject of the present invention is also a nucleic acid fragment, characterized in that its nucleotide sequence is chosen from those which are contained in one of the nucleotide sequences included in the env or gag genes of the group O HIV-1 strains and its variants and comprises:
For the purposes of the present invention, nucleic sequence is understood to mean the sequences, as specified above and their complementary sequences, as well as the sequences containing them.
Such sequences find application both in the specific identification of a group O HIV-1, as diagnostic reagent, alone or in a pool with other reagents, for the identification of any HIV-1 or alternatively, depending on the cases, as reagent for intra-group O differentiation.
These sequences may be used in particular in diagnostic tests comprising either a direct hybridization with the viral sequence to be detected, or an amplification of the said viral sequence, using, as primers, an oligonucleotide, included in any one of the above sequences and in particular one of the following sequences:
GAG/5'CAM or G5: CAGGGACAAATGGTACATCA (positions 1250-1269) (SEQ ID No. 74) PA2 GAG/3'CAM or G3: AGTAGCTTGCTCAGCTCTTAAT (positions 1768-1747) (SEQ ID No. 75) PA2 SEQ ID No. 22 (gp41/5'CAM-1): AGRGAAAAAGAGCAGTAGGAT (positions 7800-7821) PA2 SEQ ID No. 23 (gp41/5'CAM-2): TCTAAGTGCAGCAGGTAGCACTAT (positions 7843-7866) PA2 SEQ ID No. 24 (gp41/3'CAM-2): CTAAGTTGCTCAAGAGTGGTA (positions 8594-8573) PA2 SEQ ID No. 25 (gp41/3'CAM-2): GTTGCTCAAGAGGTGGTAAGT (positions 8590-8570) PA2 C2V3/5'CAM or V3L5: TRGTTACTTGTACACATGGCAT (positions 6991-7012) (SEQ ID No. 76) PA2 C2V3/3'CAM or V3L3: ACAATAAAAGAATTCTCCATGACAGT (positions 7421-7396) (SEQ ID No. 77).
The abovementioned positions correspond to those of the Ant70 sequence (Myers, Korber et al., cited above).
The subject of the present invention is also group O HIV-1 strains, characterized in that they comprise at least one of the sequences selected from the group consisting of the sequences SEQ ID No. 1 to SEQ ID No. 7 or SEQ ID No. 50 to SEQ ID No. 53, at least one of the sequences selected from the group consisting of the sequences SEQ ID No. 8 to SEQ ID No. 14 or SEQ ID No. 54 to SEQ ID No. 57 and at least one of the sequences selected from the group consisting of the sequences SEQ ID No. 15 to SEQ ID No. 21 or SEQ ID No. 58 to SEQ ID No. 61.
According to an advantageous embodiment of the said strain, it comprises the sequences SEQ ID No. 50, SEQ ID No. 54, SEQ ID No. 58; this strain has been called BCF09.
According to another advantageous embodiment of the said strain, it comprises the sequences SEQ ID No. 51, SEQ ID No. 55, SEQ ID No. 59; this strain has been called BCF12.
According to yet another advantageous embodiment of the said strain, it comprises the sequences SEQ ID No. 52, SEQ ID No. 56, SEQ ID No. 60; this strain has been called BCF13.
According to yet another advantageous embodiment of the said strain, it comprises the sequences SEQ ID No. 53, SEQ ID No. 57, SEQ ID No. 61; this strain has been called BCF14.
According to another advantageous embodiment of the said strain, it comprises the sequences SEQ ID No. 7, SEQ ID No. 14, SEQ ID No. 21; this strain has been called BCF11.
According to yet another advantageous embodiment of the said strain, it comprises the sequences SEQ ID No. 4, SEQ ID No. 11, SEQ ID No. 18; this strain has been called BCF06.
The subject of the invention is also the use of the sequences described above for carrying out a process of hybridization or gene amplification of nucleic sequences of the HIV-1 type, these processes being applicable to the in vitro diagnosis of the potential infection of an individual with an HIV-1 type virus, including group O.
This in vitro diagnostic method is carried out using a biological sample (serum, circulating lymphocytes) and comprises:
The subject of the invention is also a peptide characterized in that it is expressed by a nucleotide sequence as defined above.
Among these peptides, there may be mentioned in particular:
The subject of the invention is also immunogenic compositions comprising one or more products of translation of the nucleotide sequences according to the invention or a fragment thereof and/or at least one of the peptides as defined above.
The subject of the invention is also the antibodies directed against one or more of the peptides described above and their use for carrying out methods of in vitro diagnosis of the infection of an individual with an HIV-1 type virus, according to processes known to persons skilled in the art.
By way of illustration, such an in vitro diagnostic method according to the invention comprises bringing a biological sample collected from a patient into contact with antibodies according to the invention, and detecting, with the aid of any appropriate process, in particular with the aid of anti-labelled immunoglobulins, the immunological complexes formed between the antigens of the HIV-1 type viruses which may be present in the biological sample and the said antibodies.
The subject of the present invention is also a process for screening and typing group O HIV-1, characterized in that it comprises bringing any of the nucleotide fragments in accordance with the invention into contact with the nucleic acid of the virus to be typed and detecting the hybrid formed.
In addition to the preceding arrangements, the invention further comprises other arrangements, which will emerge from the description which follows, which refers to examples for carrying out the process which is the subject of the present invention as well as to the accompanying drawings, in which: